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An apparatus and method according to the invention combine more than one
optical modality (spectroscopic method), including but not limited to
fluorescence, absorption, reflectance, polarization anisotropy, and phase
modulation, to decouple morphological and biochemical changes associated
with tissue changes due to disease, and thus to provide an accurate
diagnosis of the tissue condition.

38. A method for diagnosing a condition of a target tissue, comprising the
steps of: a.) irradiating a target tissue with excitation electromagnetic
radiation; b.) sensing a returned electromagnetic radiation returned from
the target tissue; c.) determining characteristics of the returned
electromagnetic radiation using at least two spectroscopic methods; d.)
combining the characteristics determined by the at least two
spectroscopic methods, thereby decoupling biochemical changes from
morphological changes in the target tissue; and e.) determining a
condition of the target tissue based on the combined determined
characteristics.

39. The method of claim 38, wherein the at least two spectroscopic methods
are selected from the group consisting of absorption measurements,
scattering measurements, reflection measurements, polarization
anisotropic measurements, steady state fluorescence measurements, and
time resolved fluorescence measurements.

40. The method of claim 39, wherein the time resolved fluorescence
measurements comprise at least one of phase modulation techniques,
polarization anisotropic techniques and techniques that directly monitor
the decay profile of fluorescent emissions.

41. The method of claim 38, wherein step b.) comprises simultaneously
sensing electromagnetic radiation emitted from the target tissue in
response to the excitation electromagnetic radiation and excitation
electromagnetic radiation that is scattered from the target tissue.

42. The method of claim 41, wherein step c.) comprises making intensity
based measurements on both said electromagnetic radiation emitted from
the target tissue in response to the excitation electromagnetic radiation
and said excitation electromagnetic radiation that is scattered from the
target tissue.

43. The method of claim 38, wherein step b.) comprises sensing
electromagnetic radiation emitted from the target tissue in response to
the excitation electromagnetic radiation and then subsequently sensing
excitation electromagnetic radiation that is scattered from the target
tissue.

44. The method of claim 38, wherein step b.) comprises sensing
electromagnetic radiation returned from a plurality of interrogation
points distributed over the target tissue.

45. The method according to claim 44, further comprising a step of
dividing the target tissue into a first set of field areas, wherein step
c.) comprises determining characteristics of the returned electromagnetic
radiation in each of said first set of field areas using at least two
spectroscopic methods, step d.) comprises combining the characteristics
determined by the at least two spectroscopic methods for each of said
first set of field areas and step e.) comprises determining a condition
of the target tissue by comparing the combined determined characteristics
of each of said first set of field areas.

46. The method of claim 45, further comprising, after determining a
condition of the target tissue by comparing the combined determined
characteristics of each of said first set of field areas, re-dividing the
target tissue into a second set of field areas, different from said first
set of field areas and the determining characteristics of the returned
electromagnetic radiation in each of said second set of field areas using
at least two spectroscopic methods, combining the characteristics
determined by the at least two spectroscopic methods for each of said
second set of field areas and determining a condition of the target
tissue by comparing the combined determined characteristics of each of
said second set of field areas.

47. The method of claim 44, wherein the method is performed using an
apparatus comprising an irradiation source, a detector and a processor,
wherein the step of sensing electromagnetic radiation returned from a
plurality of interrogation points comprises the steps of: sensing
electromagnetic radiation returned from the target tissue from a first
subset of the plurality of interrogation points; moving at least one of
the apparatus and the tissue; sensing electromagnetic radiation returned
from the target tissue from a second subset of the plurality of
interrogation points; again moving at least one of the apparatus and the
tissue; and continuing this process until sensing has been peformed at
all of the plurality of interrogation points.

48. A system for determining a condition of a target tissue in a human or
animal, comprising: a electromagnetic radiation source for providing
excitation electromagnetic radiation; a device that couples the
excitation electromagnetic radiation to a target tissue; a device that
senses electromagnetic radiation returned from the target tissue; a
processor configured to determine characteristics of the returned
electromagnetic radiation using at least two spectroscopic methods,
wherein the processor combines the characteristics determined by each of
the at least two spectroscopic methods in order to decouple biochemical
changes from morphological changes in the target tissue and determines a
condition of the target tissue based on the combined determined
characteristics.

49. The system of claim 48, wherein the at least two spectroscopic methods
comprise fluorescence measurement methods and scattering or reflectance
measurement methods.

50. The system of claim 48, wherein the at least two spectroscopic methods
are selected from the group consisting of absorption measurements,
scattering measurements, reflectance measurements, polarization
anisotropy measurements, steady state fluorescence measurements and time
resolved fluorescence measurements.

51. The system of claim 48, wherein the device that senses returned
electromagnetic radiation is configured to simultaneously sense
fluorescent radiation emitted by endogenous fluorophores in response to
the excitation radiation and excitation electromagnetic radiation that is
scattered from the target tissue.

52. The system of claim 48, wherein the device that senses electromagnetic
radiation is configured to sense electromagnetic radiation returned from
a plurality of interrogation points distributed over the target tissue.

53. The system according to claim 52, wherein the processor divides the
target tissue into a first set of field areas, determines characteristics
of the returned electromagnetic radiation in each of said first set of
field areas using said at least two spectroscopic methods, combines the
characteristics determined by each of said at least two spectroscopic
methods for each of said first set of field areas and determines a
condition of the target tissue in each of said first set of field areas
based on the combined determined characteristics of the respective field
areas.

54. The system of claim 53, wherein the processor is further configured
to, after the processor determines a condition of the target tissue in
each of the first set of field areas based on the combined determined
characteristics of the respective field areas, divide the target tissue
into a second set of field areas, different from the first set of field
areas; determine characteristics of the returned electromagnetic
radiation in each of said second set of field areas using said at least
two spectroscopic methods, combine the characteristics determined by each
of said at least two spectroscopic methods for each of said second set of
field areas and determine a condition of the target tissue in each of the
second set of field areas based on the combined determined
characteristics of the respective field areas.

55. A system for determining a condition of a target tissue in a human or
animal, comprising: an electromagnetic radiation source for providing
excitation electromagnetic radiation; a device that couples the
excitation electromagnetic radiation to a target tissue; a device that
senses electromagnetic radiation returned from the target tissue; and a
processor configured to determine characteristics of the returned
electromagnetic radiation using at least two spectroscopic methods,
thereby decoupling biochemical changes from morphological changes in the
target tissue occurring due to disease and determine a condition of the
target tissue based on the determined characteristics.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to apparatus and methods for determining
tissue characteristics of, for example, a human or animal.

[0003] 2. Background of the Related Art

[0004] Spectroscopic methods for determining tissue characteristics are
known and have been widely used to interrogate changes in tissue. A
number of these distinct spectroscopic techniques are available that
provide specific information depending on the nature of the interaction
of light with cells and the natural chromophores present in tissue. These
interactions include the absorption of light at a particular wavelength,
the reemission of absorbed light as fluorescence, the scattering
(redirection) of light at a particular wavelength and the change in
polarization between the absorbed or scattered light and the reemitted
light.

[0005] For example, it is known to irradiate a target tissue with
electromagnetic radiation and to detect returned electromagnetic
radiation to determine characteristics of the target tissue. In known
methods, the amplitudes and wavelengths of the returned radiation are
analyzed to determine characteristics of the target tissue. For instance,
U.S. Pat. No. 4,718,417 to Kittrell et al. discloses a method for
diagnosing the type of tissue within an artery, wherein a catheter is
inserted into an artery and excitation light at particular wavelengths is
used to illuminate the interior wall of the artery. Material or tissue
within the artery wall emits fluorescent radiation in response to the
excitation light. A detector detects the fluorescent radiation and
analyzes the amplitudes and wavelengths of the emitted fluorescent
radiation to determine whether the illuminated portion of the artery wall
is normal, or covered with plaque. The contents of U.S. Pat. No.
4,718,417 are hereby incorporated by reference.

[0006] U.S. Pat. No. 4,930,516 to Alfano et al. discloses a method for
detecting cancerous tissue, wherein a tissue sample is illuminated with
excitation light at a first wavelength, and fluorescent radiation emitted
in response to the excitation light is detected. The wavelength and
amplitude of the emitted fluorescent radiation are then examined to
determine whether the tissue sample is cancerous or normal. Normal tissue
will typically have amplitude peaks at certain known wavelengths, whereas
cancerous tissue will have amplitude peaks at different wavelengths.
Alternatively the spectral amplitude of normal tissue will differ from
cancerous tissue at the same wavelength. The disclosure of U.S. Pat. No.
4,930,516 is hereby incorporated by reference. The above described
methods are referred to as fluorescence spectroscopy.

[0007] Still other patents, such as U.S. Pat. No. 5,369,496 to Alfano et
al., disclose methods for determining characteristics of biological
materials, wherein a target tissue is illuminated with light, and
backscattered or reflected light is analyzed to determine the tissue
characteristics. The contents of U.S. Pat. No. 5,369,496 are hereby
incorporated by reference. This type of method is referred to as
absorption spectroscopy.

[0008] It is also known to look at the decay time of fluorescent emissions
to determine the type or condition of an illuminated tissue. These
methods are referred to as time resolved spectroscopy. Generally,
apparatus for detection of the lifetime of fluorescent emissions have
concentrated on directly measuring the lifetime of the fluorescent
emissions. Typically, a very short burst of excitation light is directed
at a target tissue, and fluorescent emissions from the target tissue are
then sensed with a detector. The amplitude of the fluorescent emissions
are recorded, over time, as the fluorescent emissions decay. The
fluorescent emissions may be sensed at specific wavelengths, or over a
range of wavelengths. The amplitude decay profile, as a function of time,
is then examined to determine a property or condition of the target
tissue.

[0009] For instance, U.S. Pat. No. 5,562,100 to Kittrell et al. discloses
a method of determining tissue characteristics that includes illuminating
a target tissue with a short pulse of excitation radiation at a
particular wavelength, and detecting fluorescent radiation emitted by the
target tissue in response to the excitation radiation. In this method,
the amplitude of the emitted radiation is recorded, over time, as the
emission decays. The amplitude profile is then used to determine
characteristics of the target tissue. Similarly, U.S. Parent No.
5,467,767 to Alfano et al. also discloses a method of determining whether
a tissue sample includes cancerous cells, wherein the amplitude decay
profile of fluorescent emissions are examined. The contents of U.S. Pat.
Nos. 5,562,100 and 5,467,767 are hereby incorporated by reference.

[0010] Other U.S. patents have explained that the decay time of
fluorescent emissions can be indirectly measured utilizing phase shift or
polarization anisotropy measurements. For instance, U.S. Pat. No.
5,624,847 to Lakowicz et al. discloses a method for determining the
presence or concentration of various substances using a phase shift
method. U.S. Pat. No. 5,515,864 to Zuckerman discloses a method for
measuring the concentration of oxygen in blood utilizing a polarization
anisotropy measurement technique. Each of these methods indirectly
measure the lifetime of fluorescent emissions generated in response to
excitation radiation. The contents of U.S. Pat. Nos. 5,624,847 and
5,515,864 are hereby incorporated by reference.

[0011] None of the prior art methods discussed above alone is sufficient
to accurately measure changes in tissue characteristics. That is, as more
fully discussed below, as tissue undergoes changes from normal to, for
example, cancerous tissue, fluorescence spectroscopy becomes less
effective in determining tissue characteristics because it is less
sensitive to the morphological changes occurring, as compared to
absorption spectroscopy. Likewise, absorption spectroscopy alone is
insufficient to assess changes in tissue characteristics because it is
less sensitive to biochemical changes in tissue, as compared to
fluorescence spectroscopy.

[0012] It is known to combine two or more measurement techniques to arrive
at a more accurate ultimate determination. For example, U.S. Pat. No.
5,582,168 to Samuels et al., the contents of which are hereby
incorporated by reference, discloses an apparatus and method for
detecting changes in the lens of an eye. Samuels et al. teach measuring
both transmission or Raman or fluorescence emission, as well as
scattering, reflection or similar effects. The material under examination
is then normalized using a ratio of the fluorescence emission intensity
to the scattering or reflected intensity. However, while this method
addresses biochemical changes due to disease, it does not address
morphological changes due to disease.

[0013] Further, generally, prior art spectroscopic methods focus on tissue
characteristics at a single point or minium number of points on the
tissue. Taking measurements at just one point or a minimum number of
points can be misleading as it does not provide a sufficient sampling of
tissue area to accurately reflect the tissue's condition.

SUMMARY OF THE INVENTION

[0014] The invention focuses on providing methods and apparatus that
provide accurate measurements of changes in characteristics of tissues.
The methods and apparatus according to the invention combine more than
one optical modality (spectroscopic method), including but not limited to
fluorescence, absorption, reflectance, polarization anisotropy, and phase
modulation to decouple morphological and biochemical changes associated
with tissue changes, and thus to provide an accurate diagnosis of the
tissue's condition. The measurements taken according to the various
spectroscopic methods can be equally weighted for diagnostic purposes, or
can be weighted in various manners to produce the best diagnostic
results. For example, the results may be weighted based on
characteristics particular to the tissue subject, such as, for example,
patient ages, hormonal metabolism, mucosal viscosity, circulatory and
nervous system differences.

[0015] The invention encompasses apparatus and methods for determining
characteristics of target tissues, wherein excitation electromagnetic
radiation is used to illuminate a target tissue and electromagnetic
radiation returned from the target tissue is analyzed to determine the
characteristics of the target tissue. Some apparatus and methods
embodying the invention can be used to perform a diagnosis at or slightly
below the tissue surface of, for example, a human or animal. For
instance, methods and apparatus embodying the invention could be used to
diagnose the condition of skin, the lining of natural body lumens such as
the gastrointestinal tract, or the surfaces of organs or blood vessels.
Other apparatus and methods embodying the invention can be used to
perform a diagnosis deep within tissues of, for example, a human or
animal, where the excitation radiation has to pass through several
centimeters of tissue before it interacts with the target tissue, such as
in diagnosis of tumors and lesions deep in a breast of a human or animal.

[0016] According to a preferred embodiment of the invention, an apparatus
and method are provided which utilize fluorescence in combination with
reflectance in order to decouple the biochemical changes from the
morphological changes. The fluorescence and reflectance information may
be separately analyzed and compared, or alternatively, can be calibrated
to take into account the attenuation due to absorption and scattering.
Other combinations of spectroscopic methods besides fluorescence and
reflectance may also be appropriate.

[0017] Measurements using the various spectroscopic methods may be taken
simultaneously, or may be taken one after the other provided that a
critical timing window, defined as the time period between the
measurements, is maintained below a certain time interval.

[0018] The above described techniques are preferably used to determine
characteristics of multiple portions of a target tissue. The target
tissue may be analyzed as a whole by simultaneously taking measurements
at a plurality of interrogation points covering substantially the entire
tissue surface, or by taking measurements at only a portion of the
plurality of interrogation points covering substantially the entire
tissue surface at timing intervals until measurements have been taken at
all of the plurality of interrogation points.

[0019] Further, the target tissue can be divided into a plurality of field
areas to create a field pattern. Measurements may then be taken at a
plurality of interrogation points within each of the field areas. The
field areas may be then separately analyzed and compared in order to
diagnose a condition of the target tissue. The target tissue can then be
redivided into a different set of field areas and the field areas
analyzed and compared in order to diagnose the condition of the tissue.
The field areas may be all identically sized and/or shaped, or may have
varied sizes and/or shapes. Further, the target tissue may be redivided
into field areas of the same size and shape as the original field areas,
which then are merely repositioned, or it may be redivided into field
areas of a different size and/or shape, or of varied sizes and/or shapes.

[0020] As discussed above, techniques embodying the invention can be used
to determine the conditions of multiple portions of a target tissue, and
the determined conditions can be used to create a map of the target
tissue. Such a map could then be either displayed on a display screen, or
presented in hard copy format.

[0021] Further, the techniques can be used to feed information into a
pattern recognition algorithm, or neural network.

[0022] Additional advantages, objects, and features of the invention will
be set forth in part in the description which follows and in part will
become apparent to those having ordinary skill in the art upon
examination of the following or may be learned from practice of the
invention. The objects and advantages of the invention may be realized
and attained as particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Preferred embodiments of the invention will now be described with
reference to the following drawing figures, wherein like elements are
referred to with like reference numerals, and wherein:

[0024] FIG. 1 is a schematic diagram showing an apparatus embodying the
invention capable of performing a phase shift measurement;

[0025] FIG. 2 is a schematic diagram of an endoscope embodying the
invention;

[0026] FIGS. 3A and 3B show another embodiment of the invention;

[0027] FIGS. 4A, 4B and 4C show the end portions of various embodiments of
the invention;

[0028] FIG. 5 is a cross-sectional view of another embodiment of the
invention;

[0029] FIGS. 6A and 6B are alternative cross-sectional views of the
apparatus of FIG. 5 taken along section line 10-10;

[0030] FIGS. 7A-7D, 8 and 9 show various arrangements of optical fibers;

[0031] FIG. 10 shows another embodiment of the invention;

[0032] FIG. 11A is a schematic diagram showing another embodiment of the
invention;

[0033] FIGS. 11B-11D show how target tissue can be divided into a
plurality of field areas;

[0034] FIG. 12 shows the steps of a method embodying the invention; and

[0035] FIGS. 13-51 are graphs illustrating the results of various tests
conducted utilizing the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] In the prior art methods in the Background of the Invention
Section, the information content of the interaction of light (and
consequently the spectroscopic method used) is, generally speaking,
specific to the type of change in tissue. That is, tumorous tissue
differs from normal tissue in several ways. Tumorous tissue is generally
derived from normal tissue after the latter has undergone several
changes. These changes can be induced by various intrinsic and extrinsic
factors. These include the presence of certain inherited traits,
chromosomal mutation, virus induced malignant transformation of cells and
the mutagenic effects of UV and X-ray irradiation, to name a few.

[0037] The earliest changes that occur in the course of normal cells
becoming malignant are biochemical. One of the first changes noted is
that of increased glycolytic activity which allows tumors to grow to a
large size with decreased oxygen requirements. Invasive tumor cells
secrete type IV collegenase destroying the basement membrane barrier, a
principle component of which it Collagen IV. This allows the invading
tumor cells to pervade into the underlying stroma or connective tissue. A
number of other enzymes (e.g. cathepsins, hyaluronidases, proteoglycans
and type I, II and III collagenases weaken the extracellular matrix and
contribute to further tumor invasion. As tumors enlarge in size to beyond
1-2 mm.sup.3, the supply of oxygen and other nutrients becomes limiting.
A number of tumors have been shown to secrete tumor angiogenesis factors,
which induce the formation of blood vessels within the tumor to supply
the necessary oxygen and nutrients for sustained tumor growth.

[0038] Morphological changes appear later in the course of tumor
progression. Such changes are defined as any change in average cell size,
cell appearance, cell arrangement and the presence of non-native cells.
In addition, increased perfusion due to the effects of angiogenesis
results in an overall difference in tissue appearance. Normal tissue is
highly differentiated in cell type and arrangement. In addition, normal
cells are highly tissue specific. Tumor cells lose this tissue
specificity, as well as cell differentiation and arrangement. A marked
difference between tumor and normal cells is the change in the
cytoskeleton, the network of microtubules and microfilaments in the
cytoplasm. The cytoskeleton in normal cells is highly organized whereas
that in tumor cells is disorganized. Moreover because tumor cells are
rapidly dividing, the chromatin content in the nucleus and the nuclear
size are both higher than in normal cells.

[0039] Absorption spectroscopy is more sensitive to the morphological
changes that occur later in tumor progression. Measurements are made
either in a transmission geometry where the sample is placed between the
light source and detector, or in a reflectance geometry where the source
and detector, are on the same side. In any configuration, changes in
tissue absorption that occur between tumors and normal tissue can be
measured. For example, the increased vascularization due to angiogenesis
causes increased blood absorption. Light propagating through and
reemitted from tissue is, however, strongly affected by light scattering
interactions and does not simply depend on the absorption spectrum of
tissue chromophores. Therefore, in addition to reporting changes in
absorption, such techniques are sensitive to changes in size, structure
and arrangement of cells and cellular organelles, all of which contribute
to a change in the scattering properties of tissue. Tumor cells have
enlarged nuclei and since nuclei have a different refractive index from
that of the cell cytoplasm, they serve as efficient light scatterers. A
similar behavior is observed from other cell organelles such as, for
example, mitochondria and endoplasmic reticuli.

[0040] In absorption spectroscopy therefore, two effects, absorption and
scattering, dictate the amount of radiation measured at the detector.
Simply stated, these effects can either be additive or may tend to cancel
out each other. It is necessary, therefore, to in some way to decouple
these effects to provide an accurate measurement of tissue properties. A
number of techniques have been described in the prior art to accomplish
this. See, for example, U.S. Pat. No. 5,630,423 to Wang, et al. and the
references cited therein, which are hereby incorporated by reference. It
is now possible to obtain within reasonable accuracy the coefficients for
scattering and absorption.

[0041] A different approach to absorption spectroscopy is the use of
reflectance depolarization techniques. In this approach linearly
polarized light is directed on the tissue and the returned reflective
image is viewed through polarizers parallel and perpendicular to the
direction of polarization of the incident light. The parallel component
has sampled the surface tissue and the perpendicular component, after
sampling deeper tissue, is scattered multiple times and is consequently
depolarized. By analyzing photons that have sampled surface tissue the
absorption spectrum of this tissue independent of scattering effects can
be generated. Additionally, by modulating the extent of depolarization in
the returned radiation used for analysis, the depth of tissue
interrogated can be controlled.

[0042] Early biochemical changes are best detected by the change in
fluorescence properties of native chromophores. The principle
fluorophores present in tissue are the aromatic amino acids tyrosine,
phenylalanine and typtophan, the metabolites NAD(H) and FAD(H) and
structural proteins collagen and elastin. All of these fluorophores
possess characteristic absorption and fluorescence spectra. The
fluorescence properties of these molecules depends upon their
physicochemical environment including pH, solvation and oxidation state.
For example, the reduced form NAD(H) fluoresces while the oxidized form
does not. The reverse is true for FAD (H). The action of various
proteases secreted by tumor cells as described above, on structural
proteins, causes the fluorescent moieties (tryptophan, phenylalanine
etc.) to be exposed to a different local environment (different
salvation, viscosity and hydrophobicity) thus changing their fluorescent
characteristics.

[0043] Although biochemical changes precede the morphological changes that
occur as a result of the former, it is unrealistic to think of diseased
tissue that differs from surrounding normal tissue only in its intrinsic
biochemistry. If this were true then by simply measuring the fluorescence
one could identify and locate disease. In reality varying degrees of
morphological change accompany the biological changes. These changes
appear later in the course of tumor progression and are defined as any
change in average cell nuclei, cell size, cell appearance, cell
arrangement and the presence of non native cells. In addition, effects of
the host response such as, for example, increased perfusion from
angiogenesis results in an overall difference in tissue appearance. The
morphological changes add more complexity to the measurements by
absorbing and scattering both excitation and fluorescent light thereby
altering the true fluorescence signal. If the tumor is early, the
possibility of measurable morphological changes having occurred are low
and consequently fluorescence alone may be able to identify early tumors
from nearby normal tissue. However, once significant changes in
morphology have occurred the measurement now involves the added
complication of deconvolving or decoupling the effects of fluorescence
spectral changes from changes in fluorescence signal due to scattering
and reabsorption. For example, in the diagnosis of hyperplasia and
adenomatous polyps from normal colonic tissue, a decrease in 390 nm
fluorescence (337 nm excitation) is seen as the tissue types change from
normal as taught by Shoemacher et. al. at pages 63-78 of Lasers in Surg.
Med (12) 1992, which is hereby incorporated by reference. This could be
interpreted as a decrease in collagen fluorescence or an increase in
hemoglobin absorption. In fact, the authors show that the effect is due
to a screening of fluorescence from collagen (itself unchanged) in the
submucosal layer by the thickening mucosa in an adenoma.

[0044] Clearly, therefore simply measuring the change in fluorescence
spectral shifts or intensity changes will not be sufficient for
accurately measuring changes in tissue characteristics, and making, for
example, a fluorescence based diagnosis. It is difficult to make a
fluorescence measurement that is truly independent of the effect of
scattering and absorbance.

[0045] In order to decouple the effects of biochemical and morphological
changes, the relative degrees of which vary depending upon the extent of
tumor progression, a multimodal approach is required. Such an approach
requires a device capable of measuring both fluorescence and absorption
spectra of the area of interest. Both measurements must be made on the
same site at preferably the same time so as to ensure identical
condition.

[0046] The decoupling can be carried out in a variety of ways, which are
later discussed.

[0047] Time resolved fluorescence methods are largely independent of the
effects of scattering and absorbance. This is especially true for
diagnosis of epithelial cancer and similar conditions where the distance
traversed by light is small. Time resolved measurement measures the
fluorescence lifetime of a fluorophore. This is an intrinsic molecular
property and as such is independent of extraneous interferences such as
fluorosphore concentration (provided a measurable signal with adequate
signal to noise is present) or light source fluctuations. Such methods
have been demonstrated for transcutaneous measurements from fluorescent
implants and have been shown to be superior to steady state fluorescence
measurements. See Bambot, et al., Biosens and Bioelectronics (10) 1995 at
pages 643-652 and U.S. Pat. No. 5,628,310 to Rao, et al. which are hereby
incorporated by reference. The same tissue biochemical changes that
result in fluorescence spectral shifts and intensity changes also
generally change fluorescence lifetimes. It is commonly known that non
radiative processes that depopulate the excited state of fluorophore
cause large changes in fluorescence lifetime. Such non radiative
processes are likely the result of a changing physicochemical environment
surrounding intrinsic fluorophores in an emerging tumor.

[0048] Time resolved methods are accomplished in either the time domain or
frequency domain, the latter is also known as phase modulation
fluorimetry. Phase modulation measurements can be accomplished with
cheaper and less complex instrumentation than is used to directly measure
the decay time of fluorescence. For example, an intensity modulated light
beam may be directed upon the sample. The fluorescence returned from the
sample is also intensity modulated at the same frequency. However,
because of the finite fluorescence lifetime of the fluorophore in tissue,
the returned fluorescence signal is phase shifted and this phase shift is
related to the fluorescence lifetime.

[0049] The biggest impediment to using time resolved methods presently is
cost. This cost is proportional to the magnitude of both the modulation
frequency and the frequency of light. The modulation frequency used is
nominally the inverse of the lifetime of the fluorosphore being
interrogated. Given the short (few nanoseconds) fluorescence lifetimes of
intrinsic chromophores in tissue that serve as markers for disease, high
modulation frequencies (several hundred megahertz) are required,
necessitating the need for RF equipment and techniques. In addition, most
intrinsic chromophores have absorption maxima at low wavelengths (high
frequencies). Solid state light sources and detectors that operate at
these wavelengths and that are capable of being intrinsically modulated
at the requisite modulation frequencies are expensive and rare. Having
said this, both areas, low wavelength light sources/detectors and RF
frequency digital electronics are an active area of research and
development and significant cost reductions are expected in the future.

[0050] An alternative to the phase change method discussed above to
determine fluorescence lifetime is the measurement of fluorescence
depolarization or anisotropy. The instrumentation used is similar to that
used for reflectance depolarization. Indeed the same instrument can
readily be used for measurement based on both principles. In clear
solution (where photons are not depolarized due to scattering the
measurement of fluorescence polarization anisotropy provides an estimate
of the fluorescence lifetime of the fluorophores being interrogated. This
is represented by the Perrin Equation (Perrin et. al.) which relates
fluorescence Anisotropy (r) to Lifetime (.tau.) r o r = 1 + r
.PHI. Equation .times. .times. 1 where r.sub.o, is the
anisotropy of the molecule when Brownian motion is absent, i.e. in the
frozen state or in a highly viscous medium, r is the time averaged
anisotropy observed, .tau. is the fluorescence lifetime of the molecule
and .phi. is the Brownian rotation correlation time.

[0051] Strictly speaking the above equation is valid only for a single
exponential decay in both fluorescence lifetime and anisotropy. The
anisotropy decay is single exponential only for a spherical molecule
(isotropic depolarization). The rotational correlation time is defined,
for simplicity, for a sphere to be; .PHI. = .eta. .times.
.times. V RT Equation .times. .times. 2 where .eta. is the
viscosity, V the volume, R the universal gas constant and T the absolute
temperature.

[0052] As illustrated in Equation 1, the anisotropy reflects changes from
both fluorescence lifetime and rotational correlation time. The
fluorescence lifetime of intrinsic fluorophores change with tumor
progression. Similarly a change in local physical properties such as
microviscosity, temperature or membrane fluidity will change the
rotational correlation time and resulting in a change in an isotropy.
U.S. Pat. Nos. 4,115,699, 4,122,348 and 4,131,800, which are hereby
incorporated by reference, disclose the measurement of changes in local
microviscosity and fluidity due to malignancy using exogenous lipophilic
dyes and the method of fluorescence depolarization.

[0053] The principle drawback with this technique when applied to in vivo
tissue measurements is the depolarization caused by multiple scattering
events in tissue. It has been shown, however, that a significant portion
of the polarized excitation remains polarized before exciting the
fluorophore and the resulting fluorescence is also substantially
polarized when it reaches the detector. Nevertheless, it is necessary to
decouple depolarization due to scattering from depolarization due to
fluorescence lifetime and rotation correlation time.

[0054] The techniques according to the invention are designed to
discriminate normal tissue from various cancerous tissue stages based on
spectroscopic data alone. Additional factors, such as, for example,
patient age, menopausal status, menstrual state, and/or previous history
of disease can be added to the spectroscopic inputs in achieving better
discrimination.

[0055] The multimodal approach according to the invention may be carried
out in an imaging mode. In other words the multiple spectroscopic methods
are used in interrogating tissue at several interrogation points at high
spatial resolution concurrently. The reasoning behind this approach is
the variability in spectroscopic signature of known normal tissue between
patients and the fact that in 99% of the patients the entire organ is not
diseased. The best way to do this without an a prior knowledge of what is
normal is to measure both the normal and abnormal tissue, that is, the
entire organ.

[0056] Visually normal areas on an organ with precancers or cancers are
always suspect and a conclusive analysis is almost always the result of a
biopsy and histology. This observation is consistent with the phenomenon
of field cancerization, as discussed by D. P. Slaughter, et al., in
"Field Cancerization in Oral Squamour Epithelium: Clinical Implication of
Multicentric Origin" in Cancer 6, 1953, at pages 963-968, which is hereby
incorporated by reference. A considerable body of evidence exists,
particulary for breast cancer, which shows that supposedly normal breast
epithelium derived from patient's breast cancer is "condemned" in that it
is precancerous. This explains the relatively high rate of second breast
cancer incident in women treated for the disease, as discussed by G. F.
Schwartz, et al. in "The Prevalence of carcinoma In Situ In Normal and
Cancer Associated Breasts", Hum Pathol 16, 1985, at pages 796-807, which
is hereby incorporated by reference. The inventors believe a similar
pattern exists with cervical cancer and may explain the high number of
women (50%) with a history of negative pap tests who develop cervical
cancer, as discussed in Cancer Diagnostics, The World Market, Clinica
Reports, PJB Publications, 1997, at page 72, which is hereby incorporated
by reference. Such a pattern further warrants the use of imaging modes in
cancer detection.

[0057] The invention will now be further discussed with reference to the
drawings.

[0058] FIG. 1 is a schematic diagram of an apparatus according to a
preferred embodiment of the invention. The apparatus includes a source
20, which produces electromagnetic radiation that is conducted to a
target tissue 50, preferably by one or more emission optical fibers 52.
The apparatus may also include a filter 22 for selectively controlling
the electromagnetic radiation emitted from the radiation source 20. The
source 20 could comprise, for example, a laser, a light emitting diode, a
fluorescent tube, an incandescent bulb, or any other type of device that
is capable of emitting electromagnetic radiation, as is well known to
those skilled in the art.

[0059] Electromagnetic radiation returned from target tissue 50, is sensed
by a detector 56. As discussed below, the detector may employ any of the
known methods for determining tissue characteristics, including but not
limited to fluorescence, absorption, reflectance, anisotropy, phase
change, and any other know spectroscopic methods including those methods
discussed in the Background of the Invention section of this disclosure.
Preferably, the detector employs two or more spectroscopic methods which
provides for a better or more accurate measure of target tissue
characteristics than one measurement alone, and thus a more complete
diagnosis of the tissue's condition.

[0060] The returned electromagnetic radiation comprises both fluorescent
emissions from fluorophores in the target tissue that have been excited
by the excitation radiation and the excitation electromagnetic radiation
that is scattered or reflected from the target tissue. In a preferred
embodiment of the invention, as later discussed, the detector 56 makes
intensity based measurements on both forms of said electromagnetic
radiation. These measurements are combined to decouple the morphological
changes from the biochemical changes. The detector may comprise, for
example, a photomultiplier tube, a photosensitive diode, a charge coupled
device, or any other type of electromagnetic radiation sensor, as is also
well known to those skilled in the art.

[0061] If the detector is a small charge coupled device, it could be
located at a distal end of an endoscope or catheter instrument. In this
instance, the charge coupled device would already be located adjacent the
target tissue such that the detector could directly sense the return
radiation. The charge coupled device would then need some means for
communicating its information to a processor 44.

[0062] If the detector is not a charge coupled device located at a distal
end of an instrument, the returned electromagnetic radiation may be
conducted to the detector 56 through one or more return optical fibers
54. The return optical fibers 54 and the excitation optical fibers 52 may
be co-located within the same instrument, or they may be located in
separate instruments. Alternately, the same optical fibers within an
instrument may be used to perform both excitation and return functions.

[0063] The processor device 44 may include a memory 45 and a display 47.
In fact, the processor device may comprise a typical personal computer.

[0064] In the preferred embodiments of the invention, the detector 56 may
detect the fluorescent emissions from fluorophores in the target tissue
simultaneously with the excitation electromagnetic radiation that is
scattered or reflected from the target tissue to provide a complete
analysis of the subject tissue. Alternatively, the device may be
configured to first detect the fluorescent emissions from fluorophores in
the target tissue, and then subsequently, the excitation electromagnetic
radiation that is scattered or reflected from the target tissue. In the
later case, the time period between detections, hereinafter referred to
as the "critical timing window," must be minimized to avoid motion
artifacts and/or significant tissue changes that will denigrate the
overall results. The time period between detections is preferably less
than approximately 0.25 seconds; however, the smaller the time period,
the more accurate the results will be.

[0065] FIG. 2 shows an endoscope that could be used to practice the
measuring techniques according to the invention. The endoscope 60
includes a transmit optical fiber bundle 52, which can convey excitation
electromagnetic radiation from a radiation source 20 to a target tissue.
The endoscope 60 also includes a return optical fiber bundle 54 for
communicating fluorescent emissions and/or reflected/scattered
electromagnetic radiation from a target tissue to a detector 56. In
alternative embodiments, the transmit and return optical fibers may be
co-located, may be the same fibers, or may be a double set of fibers, as
discussed below.

[0066] That is, it is preferable to make simultaneous detections at a
plurality of interrogation points rather than at just one point or a
minimum number of points. This allows evaluation of the field effect
changes over an area of the tissue or substantially the entire tissue, as
will be more fully discussed below. Taking measurements at just one
interrogation point or a minimum number of interrogation points can be
misleading as it does not provide a sufficient sampling of tissue area to
accurately reflect the tissue's condition.

[0067] For example, the detector could be configured to make detections at
a large number of interrogation points distributed over substantially the
entire surface area of the subject tissue. That is, return optical fibers
54 could include a large number of optical fibers distributed to allow
detections to be made at a corresponding large number of interrogation
points on the tissue, preferably covering substantially the entire
surface of the subject tissue. Each of the optical fibers could transmit
excitation electromagnetic radiation to the subject tissue and then
return the return electromagnetic radiation to the detector 56. The
tissue could be analyzed as a whole, or divided into a plurality of field
areas.

[0068] Alternatively, a transmitting optical fiber and a return optical
fiber could be located at each of the interrogation points (see, for
example, FIG. 7B). Further, each interrogation point could include a
double set of optical fibers, a transmitting optical fiber and a return
optical fiber for detecting fluorescence, and a transmitting optical
fiber and a return optical fiber for detecting scattering or reflectance
(see, for example, FIG. 7C). In such a case, the optical fibers could be
arranged to focus on the same point on the subject tissue (see, for
example, FIG. 7D).

[0069] Additionally, the apparatus may include a rotatable core 114, as
discussed with respect to the embodiment of FIG. 5, or alternatively, the
tissue may be mounted on a rotatable table (not shown), so that the
detector 56 would make detections at just a portion of the multiple
interrogation points. Then, either the rotatable head or the rotatable
table could be rotated and the detector would make detections at the next
set of interrogation points. The process would continue to complete, for
example, six rotations in order to cover substantially the entire surface
of the subject tissue.

[0070] The endoscope 60 may also include a handle 62 for positioning the
endoscope, or for operating a device 64 on a distal end of the endoscope
60 intended to remove tissue samples from a patient. The endoscope may
also include a device 66 for introducing a dose of medication to a target
tissue. Also, the source of electromagnetic radiation 20 may be
configured to emit a burst of therapeutic radiation that could be
delivered to a target tissue by the endoscope.

[0071] FIGS. 3A and 3B show the structure of an endoscope or catheter
which may embody the invention. The apparatus includes a long body
portion 70 which is intended to be inserted into a body of a human or
animal. The body portion 70 must have a diameter sufficiently small to be
inserted into blood vessels or other natural lumens of the human or
animal.

[0072] The device includes a proximal end 80, which holds proximal ends of
optical fibers 72a-72c. The optical fibers extend down the length of the
device and terminate at a distal holding portion 74. The distal holding
portion 74 holds the optical fibers in a predetermined orientation. The
optical fibers are held such that they can illuminate selected portions
of the distal end 76 of the device. This orientation also allows the
distal end of the optical fibers to receive radiation from selected areas
outside the distal end 76 of the device.

[0073] As best seen in FIG. 3B, the optical fibers are arranged such that
there is a single central optical fiber 72a surrounded by a first ring of
optical fibers 72b, which is in turn surrounded by a second ring of
optical fibers 72c. Of course, other orientations of the optical fibers
are possible.

[0074] By applying excitation electromagnetic radiation to selected ones
of the optical fibers, and monitoring the returned electromagnetic
radiation through selected ones of the optical fibers, it is possible to
determine characteristics of target tissues at selected locations outside
the distal end of the device. For instance, if the central optical fiber
72a emits electromagnetic radiation 90 toward a target tissue, and
returned electromagnetic radiation is sensed through the same optical
fiber, the returned electromagnetic radiation can be analyzed using any
of the above methods to determine characteristics of a target tissue
located adjacent the center of the distal end of the device. The same
process can be used to determine the condition of a target tissue at
different locations around the distal end of the device.

[0075] FIGS. 4A-4C show various different distal ends of the device.

[0076] In FIG. 4A, the distal ends of the optical fibers are held by a
holding portion 98 that aims the distal ends of the optical fibers 97 in
a particular direction. A flexible wire or bar 96 is attached to the
holding portion 98 and extends to the proximal end of the device. By
rotating the flexible wire or bar 96, the holding portion 98 can also be
rotated. This allows the distal ends of the optical fibers to be aimed at
different portions of the distal end of the device.

[0077] FIG. 4B shows another embodiment of the invention that includes one
or more inflatable balloon portions 92a, 92b. An optical fiber 72 is
located in the center of the device by a holding portion 94. Each of the
inflatable balloons 92a, 92b is also held by the holding portion 94. By
selectively inflating or deflating the different balloon portions, the
optical fiber 72 may be aimed to illuminate different portions of the
distal end of the device or to receive return radiation from selected
locations adjacent the distal end of the device.

[0078] FIG. 4C shows an embodiment of the device similar to the embodiment
shown in FIGS. 3A and 3B. This figure shows how electromagnetic radiation
passing down through the optical fibers 72a-72c can be used to
selectively illuminate material or tissue adjacent selected portions of
the distal end of the device. In FIG. 4C, only the upper optical fibers
are emitting electromagnetic radiation outside the device. This
electromagnetic radiation is being used to destroy or atomize plaque
which has formed on an inner wall of a blood vessel. By applying
electromagnetic radiation to selected ones of the optical fibers, a
doctor can carefully remove or correct problems with target tissues or
materials.

[0079] Another device embodying the invention that can be used to
determine tissue characteristics is shown, in longitudinal cross-section,
in FIG. 5. The instrument 110 includes a cylindrical outer housing 112
with a circular end cap 120 configured to abut the target tissue. A
rotating cylindrical inner core 114 is mounted in the outer housing 112.
A bundle of optical fibers 116 are located inside the inner core 114.

[0080] The optical fibers 116 pass down the length of the inner core 114
and are arranged in a specific pattern at the end adjacent the end cap
120 of the outer housing 112. The end of the inner core 114 adjacent the
end cap 120 is mounted within the outer housing 112 with a rotating
bearing 122. The end cap 120 is at least partially transparent or
transmissive so that electromagnetic radiation can pass from the optical
fibers, through the end cap, to illuminate a target tissue adjacent the
end cap 120. Light scattered from or generated by the target tissue would
then pass back through the end cap 120 and back down the optical fibers
116.

[0081] The inner core 114 is also mounted inside the outer housing 112 by
a detent mechanism 118. The detent mechanism is intended to support the
inner core 114, and ensure that the inner core is rotatable within the
outer housing 112 by predetermined angular amounts.

[0082] A cross sectional view of an embodiment of the instrument, taken
along section line 10-10 of FIG. 5, is shown in FIG. 6A. The inner core
114 is supported within the outer housing 112 by the detent mechanism. In
this embodiment, the detent mechanism includes two mounts 134 with spring
loaded fingers 136 that are biased away from the inner core 114. The
detent mechanism also includes four stoppers 130, each of which has a
central depression 132. The spring loaded fingers 136 are configured to
engage the central depressions 132 of the stoppers 130 to cause the
rotatable inner core to come to rest at predetermined angular rotational
positions. In the embodiment shown in FIG. 6A, four stoppers are provided
in the inner surface of the outer housing 112. Thus, the inner core 114
will be rotatable in increments of approximately 90.degree.. In alternate
embodiments similar to the one shown in FIG. 6A, four mounts 134, each
having its own spring loaded finger 136, could be attached to the inner
core 114. The provision of four such mounts would serve to keep the inner
core 114 better centered inside the outer housing 112.

[0083] An alternate embodiment of the detent mechanism is shown in FIG.
6B. In this embodiment, six stoppers 130 are spaced around the inside of
the outer housing 112. Three mounts 134, each having its own spring
loaded finger 136, are mounted on the inner core 114. The three mounts
134 are spaced around the exterior of the inner core 114 approximately
120.degree. apart. This embodiment will allow the inner core to be
rotated to predetermined positions in increments of 60.degree.. In
addition, the location of the three mounts, 120.degree. apart, helps to
keep the inner core 114 supported in the center of the outer housing 112.

[0084] With reference to FIG. 5, the ends of the optical fibers may be
mounted on a circular end plate 121 that holds the optical fibers in a
predetermined pattern. The circular end plate 121 would be rigidly
attached to the end of the cylindrical inner core 114. In addition, an
index matching agent 123 may be located between the end plate 121 and the
end cap 120 on the outer housing 112. The index matching agent 123 can
serve as both an optical index matching agent, and as a lubricant to
allow free rotation of the end plate 121 relative to the end cap 120.

[0085] A diagram showing how the optical fibers are positioned on the face
of an embodiment of the instrument is shown in FIG. 7A. The face of the
instrument, which would be the end cap 120 of the device shown in FIG. 5,
is indicated by reference number 140 in FIG. 7A. The black circles 142
represent the locations of optical fibers behind the end cap 120. The
hollow circles 144 represent the positions that the optical fibers will
move to if the inner core 114 of the instrument is rotated approximately
90.degree.. Thus, each of the circles represent positions that can be
interrogated with the optical fibers.

[0086] In some embodiments of the device, a single optical fiber will be
located at each of the positions shown by the black circles 142 in FIG.
7A. In this instance, excitation light would travel down the fiber and be
emitted at each interrogation position indicated by a black circle 142.
Light scattered from or produced by the target tissue would travel back
up the same fibers to a detector or detector array, such as detector 56
shown in FIG. 1.

[0087] In alternate embodiments, pairs of optical fibers could be located
at each position indicated by the black circles 142A, 142B, as shown in
FIG. 7B. In the alternate embodiments, one optical fiber of each pair
would conduct excitation light to the target tissue, and the second
optical fiber of each pair would conduct light scattered from or
generated by the target tissue to a detector. In still other alternate
embodiments, multiple fibers for carrying excitation light and/or
multiple fibers for carrying light scattered from or generated by the
target tissue could be located at each interrogation position indicated
by a black circles 142A, 142B, 142C, 142D to allow simultaneous detection
of, for example, both fluorescence and reflectance, as shown in FIG. 7C.
In this latter case, the optical fibers could be arranged to focus on the
same point of subject tissue, as shown in FIG. 7D.

[0088] To use an instrument having the optical fiber pattern shown in FIG.
7A, the instrument would first be positioned so that the end cap 120 is
adjacent the target tissue. The end cap 120 may be in contact with the
target tissue, or it might be spaced from the surface of the target
tissue. Also, an index matching material may be interposed between the
end cap 120 and the target tissue. Then, the optical fibers would be used
during a first measurement cycle to simultaneously measure tissue
characteristics at each of the interrogation positions in FIG. 7A having
a black circle 142. The tissue characteristics could be measured using
any of the measurement techniques discussed above. Then, the inner core
114 would be rotated approximately 90.degree. within the outer housing
112, and the optical fibers would be used during a second measurement
cycle to simultaneously measure tissue characteristics at each of the
interrogation positions in FIG. 7 having a hollow circle 144.

[0089] The instrument may include markings (not shown) on the end cap 120
or elsewhere, which acts as a locator tool to allow a user to determine
how many rotations have been made, and thus how much of the tissue has
been analyzed.

[0090] Constructing an instrument as shown in FIGS. 5, 6A or 6B, and
having any of the optical fiber patterns shown in FIGS. 7A-7D, has many
important advantages. For example, constructing an instrument in this
manner allows the instrument to interrogate many more points in the
target tissue than would have been possible if the inner core did not
rotate. The ability to rotate the inner core 114, and take a second
series of measurements at different locations on the target tissue,
essentially increases the resolution of the device.

[0091] In addition, when a large number of optical fibers are packed into
the tissue contacting face of an instrument, cross-talk between the
optical fibers can occur. The cross-talk can occur when excitation light
from one interrogation position scatters from the target tissue and
enters an adjacent interrogation position. Cross-talk can also occur if
excitation light from a first interrogation position travels through the
target tissue and enters an adjacent interrogation position. One of the
easiest ways to reduce or eliminate cross-talk is to space the
interrogation positions farther apart. However, increasing the spacing
between interrogation positions will reduce the resolution of the device.

[0092] An instrument embodying the invention, with a rotatable inner core,
allows the interrogation positions during any single measurement cycle to
be spaced far enough apart to reduce or substantially eliminate
cross-talk. Because multiple measurement cycles are used, the device is
able to obtain excellent resolution. Thus, good resolution is obtained
without the negative impact to sensitivity or selectivity caused by
cross-talk. In addition, fewer optical fibers and fewer corresponding
detectors are required to obtain a given resolution.

[0093] In addition, the ability to obtain a plurality of tissue
measurements simultaneously from positions spaced across the entire
target tissue has other benefits. If the instrument is intended to detect
cancerous growths or other tissue maladies, the target tissue area
interrogated by the instrument is likely to have both normal tissue, and
diseased tissue. As noted above, tissue characteristics can vary
significantly from person to person, and the tissue characteristics can
vary significantly over relatively short periods of time. For these
reasons, one way to determine the locations of diseased areas is to
establish a baseline for normal tissue, then compare the measurement
results for each interrogation point to the baseline measurement. The
easiest way to determine the location of a diseased area is to simply
look for a measurement aberration or variance.

[0094] Because tissue characteristics can change relatively quickly, in
order to establish accurate, clearly defined variances between tissue
characteristics, it is desirable to take a plurality of readings
simultaneously over as large an area as possible. In the preferred method
according to the invention, this could include taking fluorescence
measurements at a plurality of interrogation points, and then
subsequently taking reflectance measurements, at the same plurality of
interrogation points. Alternately, the fluorescence and reflectance
measurements could be taken simultaneously. Ideally, all measurements
should be conducted during the same time period. In a preferred
embodiment, the apparatus and method conduct measurements at least within
this critical time window. The critical time window is defined as the
maximum duration of time between two spectroscopic measurements which
yields the benefits described herein. Although this value may vary
depending on a variety of factors including those described below, it has
been determined that the critical timing window between subsequent
measurements should be less than approximately 0.25 seconds and more
preferably less than approximately 0.1 second, as further discussed
below.

[0095] There are several effects which make it desirable to conduct
fluorescent and reflectance measurements of the interrogated points
either simultaneously, or as nearly simultaneously as possible. First,
changes in blood pressure, which occur during each heart beat cycle can
have a large affect on blood content in the tissue. Because blood
strongly absorbs certain wavelengths of light, the varying amount of
blood present at an interrogated point during different parts of the
heart beat cycle can cause significantly varying measurement results.

[0096] To eliminate this potential error source, both fluorescent and
reflectance measurements should be taken within a small enough time
window that the blood content remains the same. Time periods of less than
approximately 0.25 seconds should be sufficient. Another way to eliminate
the potential error is to take multiple measurements of the same
interrogation point during different portions of the heart beat cycle,
then average the results.

[0097] Another factor to consider is patient movement. If the patient
moves, even slightly, during a measurement cycle, the contact pressure
between the measurement instrument and the interrogated tissue can
change. This can also affect the measurement results. Thus, obtaining
measurements simultaneously, or as nearly simultaneously as possible,
also helps to prevent measurement errors caused by patient movement.

[0098] Also, because tissue tumors can be as small as approximately 1 mm,
the resolution of the device is preferably approximately 1 mm. In other
words, to obtain the requisite resolution, the spacing between
interrogation positions should be approximately 1 mm. Unfortunately, when
the interrogation positions are approximately 1 mm apart during a single
measurement cycle, significant cross-talk can occur, and the accuracy of
the measurement results is poor.

[0099] An instrument embodying the invention allows the interrogation
positions to be spaced sufficiently far apart to essentially eliminate
cross-talk, while still obtaining the requisite 1 mm resolution. Although
not all measurements are obtained at exactly the same time, during each
measurement cycle, simultaneous measurements are made at positions spaced
across the entire target tissue, which should include both normal and
diseased areas. Thus, the results from each measurement cycle can be used
to detect variances in tissue characteristics that help to localize
diseased areas. For these reasons, an instrument embodying the invention
balances the competing design requirements of resolution, elimination of
cross-talk, and the desire to make all measurements simultaneously to
ensure that time-varying tissue characteristics are taken into account.

[0100] A second arrangement for the optical fibers of a device as shown in
FIG. 5 is depicted in FIG. 8. In this embodiment, the interrogation
positions are arranged in a hexagonal honeycomb pattern. The black
circles 142 indicate the positions that would be occupied by optical
fibers during a first measurement cycle, and the hollow circles 144
indicate positions that would be occupied by the optical fibers during a
second measurement cycle after the inner core 112 has been rotated by
approximately 60.degree.. This pattern achieves maximum spacing between
adjacent interrogation positions during each measurement cycle, and
essentially doubles the resolution of the instrument.

[0101] A third arrangement for the optical fibers of a device shown in
FIG. 5 is depicted in FIG. 9. In this embodiment, the optical fibers are
again arranged according to a hexagonal honeycomb pattern. However, far
fewer optical fibers are used in this embodiment. This third embodiment
is intended for use in a measurement process that calls for six
measurement cycles. The inner core of the device would be rotated
approximately 60.degree. between each measurement cycle. Over the course
of the six measurement cycles, the device would ultimately interrogate
all the black circled 142 and hollow circled 144 interrogation positions
shown in FIG. 9. This embodiment allows for even greater separation
distances between the interrogation positions during a single measurement
cycle (to reduce or substantially eliminate cross-talk), while still
achieving excellent measurement resolution. In addition, far fewer
optical fibers and corresponding detectors would be required to achieve a
given measurement resolution.

[0102] Experimental studies were conducted by the applicants to determine
the spacing between interrogation positions that is needed to
substantially eliminate cross-talk. The studies were conducted using a
pair of optical fibers at each interrogation position, wherein one fiber
in each pair provides excitation light, and the other fiber in each pair
is used to detect light. The excitation optical fibers had a diameter of
approximately 200 .mu.m, the detection fibers had a diameter of
approximately 100 .mu.m. Measurements were made on optical reference
standards, and tissue. Under these conditions, it was necessary to space
the interrogation positions approximately 3 mm apart to substantially
eliminate cross-talk. Thus, if an instrument were not designed as
described above, so that the inner core can rotate the interrogation
positions to different locations on the target tissue, the device would
only be capable of achieving a resolution of approximately 3 mm.

[0103] The presently preferred embodiment of the invention utilizes an
optical fiber pattern similar to the one shown in FIG. 9. Thus, the
device is designed to conduct six measurement cycles to complete all
measurements within the target tissue. The inner core 114 is rotated
60.degree. between each measurement cycle. The presently preferred
embodiment utilizes optical fiber pairs at each interrogation position.
Each optical fiber pair includes an excitation fiber having an
approximately 200 .mu.m diameter, and a detection optical fiber having an
approximately 100 .mu.m diameter. The arrangement of the optical fibers
allows the interrogation positions to be spaced approximately 3.0-3.5 mm
apart, while still achieving a resolution of approximately 1 mm.

[0104] To determine the locations of diseased areas within a target tissue
it is necessary to take measurements at a plurality of different
locations in the target tissue spaced in at least two dimensions. Each
measurement may require multiple excitation wavelengths, and detection of
multiple wavelengths of scattered or generated light. Thus, the
measurements involve three measurement dimensions, two dimensions for the
area of the target tissue, and a third dimension comprising the spectral
information. A device capable of conducting measurements in these three
dimensions is shown in FIG. 10.

[0105] The instrument includes a light source 20, and a filter assembly
22. A plurality of excitation optical fibers 116a lead from the filter
assembly 22 to the target tissue 50. A plurality of detection fibers 116b
lead away from the target tissue 50. The excitation optical fibers 116a
and the detection optical fibers 116b are arranged in pairs as described
above.

[0106] The light source 20 and filter assembly 22 allow specific
wavelengths of light to be used to illuminate the target tissue 50 via
the excitation optical fibers 116a. The filter assembly 22 could be a
single band pass optical filter, or multiple optical filters that can be
selectively placed between the light source 20 and the excitation optical
fibers 116a. Alternatively, the light source 20 and filter assembly 22
could be replaced with a wavelength tunable light source. In yet other
alternate embodiments, a plurality of light sources, such as lasers,
could be used to selectively output specific wavelengths or wavelength
bands of excitation light. Other sources may also be appropriate.

[0107] The detection fibers lead to an optical system 55. The light from
the detection fibers 116b passes through the optical system and into a
detector array 56. The detector array may comprise a plurality of
photosensitive detectors, or a plurality of spectrophotometers. The
detector array 56 is preferably able to obtain measurement results for
each of the detection fibers 116b simultaneously.

[0108] The optical system 55 can include a plurality of optical filters
that allow the detector array 56 to determine the intensity of light at
certain predetermined wavelengths. In a preferred embodiment, the
detector array would be a two dimensional array of photosensitive
detectors, such as a charge coupled device (CCD). The optical system
would comprise a spectrograph that is configured to separate the light
from each detection optical fiber 116b into a plurality of different
wavelengths, and to focus the different wavelengths across a line of
pixels on the CCD. Thus, each line of pixels on the CCD would correspond
to a single detection fiber. The intensities of the different wavelengths
of light carried by a single detection fiber 116b could be determined
based on the outputs of a line pixels of the CCD. The greater the output
of a particular pixel, the greater the intensity at a particular
wavelength.

[0109] The preferred embodiment is able to achieve excellent flexibility.
Because all wavelengths of light are always detected, the instrument
software can simply select the pixels of interest for each measurement,
and thereby determine the intensity at particular wavelengths. During a
first measurement, certain pixels representative of fluorescent
characteristics could be examined. During a subsequent measurement,
different pixels representative of scattering characteristics could be
examined. Also, the device could be essentially re-configured to take
completely different measurements by simply changing the control
software. Thus, a single device could be used for a wide variety of
different kinds of measurements.

[0110] In preferred methods of the invention, one of the structures
described above would be used to conduct a series of measurements cycles.
Where the embodiment having the rotatable core is employed, the inner
core of the device would be rotated between measurement cycles. Once all
measurements of a measurement cycle are completed, the inner core would
be rotated, and additional measurement cycles would be conducted.

[0111] In the preferred methods, however, measurements are conducted using
two or more spectroscopic methods during each measurement cycle. For
instance, during a single measurement cycle the device may conduct a
measurement of fluorescent characteristics, and a measurement of
reflectance characteristics. However, other measurements and combinations
of spectroscopic methods may also be appropriate. Then, the fluorescence
and reflectance measurements can be compared and analyzed to decouple the
effects due to biochemical and morphological tissue changes to provide
for a more accurate diagnosis of the tissue's conditions.

[0112] As previously discussed, the measurements can be taken over
substantially the entire surface area of the subject tissue,
simultaneously or in intervals, and the results analyzed. Alternatively,
the subject tissue can be divided into field areas to create a field
pattern. Dividing the subject tissue into field areas allows analysis of
particular areas of the tissue, for example, particular areas of the
tissue where changes are likely to occur.

[0113] For example, the apparatus of FIG. 1 could further include a field
area adjusting unit 560 and field area processing unit 570, as shown in
FIG. 11A. The field area adjusting unit 560 would divide the target
tissue into a plurality of field areas 580, as shown in FIGS. 11B-11D, to
create a field pattern 500. The field areas 580 could be any desired
shape and size (see, for example, the different sized and shaped field
areas shown in FIGS. 11B-11D). Further, the divisions could be based on
visual inspection of the target tissue, or on results of previous testing
performed on the target tissue, and could be preprogramed into the
apparatus, or input by a user. Measurements would then be taken by the
detector 54 at each of a plurality of interrogation points 542 within the
respective field area and the field area processing unit 570 would then
analyze the measurements for each of the respective field areas 580. The
field area processing unit 570 could further compare the results for each
respective field area 580 to the results for other field areas 580.

[0114] FIGS. 11B and 11C show 4 and 8 "pie-shaped" field areas,
respectively. In each case, after measurements were taken by the detector
54 at each of a plurality of interrogation points 542 within the
respective field areas and the results analyzed by the field area
processing unit 570 for each of the respective field areas 580, the field
area adjusting unit 560 could reset the field areas 580 by rotating the
field area to group different sets of interrogation points (see arrow in
FIG. 11C), or could set field areas having a different size and shape,
such as the field areas shown in FIG. 11D. As shown in FIG. 11 D, these
field areas do not need to be identical in size and/or shape.

[0115] Alternatively, the field area adjusting unit 560 and field area
processing unit 570 could be incorporated into the processor 44 and the
divisions could be preprogramed into the processor or accompanying
software.

[0116] FIG. 12 shows steps of a preferred method according to the
invention. In a first step S1000, a target tissue is illuminated with
electromagnetic radiation at predetermined wavelengths, preferably one
wavelength for detecting fluorescence characteristics and one wavelength
for detecting reflectance characteristics. In a second step S1010, the
detector 56, preferably utilizing one of the optical fiber arrangements
discussed above, detects returned electromagnetic radiation. In step
S1020, the fluorescence and reflectance intensities are calculated, and
in step S1030, the fluorescence and reflectance intensities are compared
and analyzed using a preferred method discussed below. In step S1040, the
tissue characteristics are determined.

[0117] The deconvolution, or decoupling can be carried out in a variety of
ways as described below. Any or all of the discriminant parameters can be
combined together in order to improve the overall discrimination.

[0118] 1. Using a linear combination of fluorescence and reflectance
measured intensities as the discriminant parameter.
P=aF.sub..lamda.m+bR.sub..lamda.x+cR.sub..lamda.m Equation 1 Where
.lamda.x is the fluorescence excitation wavelength, .lamda.m is the
fluorescence emission wavelength, F is the fluorescence intensity and R
is the reflectance intensity. The factors a, b and c are weighing factors
that are empirically selected to give the best discrimination.

[0121] Where .lamda.1m and .lamda.2m are two distinct fluorescence
emission wavelengths, .lamda.1x and .lamda.2x are the corresponding
excitation wavelengths, F is the fluorescence intensity and R is the
reflectance intensity. The factors a, b and c are weighing factors that
are empirically selected to give the best discrimination.

[0122] 4. Using quantum yield (also known as quantum efficiency)
measurement as the discriminant parameter. The quantum yield defines the
true fluorescence yield in terms of the number of fluorescence photons
generated by the fluorophore per photon of light absorbed. P =
aF _ .lamda. .times. .times. m .times. 1 - bR
.lamda. .times. .times. x Equation .times. .times. 4
The fluorescence and reflectance intensities are corrected for background
light and normalized to the intensities measured off a calibration
target. The factors a and b are weighing factors that are empirically
selected to give the best discrimination.

[0123] 5. Blood has broadband absorbance with three distinct visible peaks
at around 410 nm, 545 nm and 575 nm. On the one hand, blood absorbance
changes from increased vascularization in cancer tissue, and is an
important marker for disease. On the other hand, blood absorbance related
artifacts occur in the measured spectra from local bleeding and
inflammation. The spectral discriminant factor described above must
therefore be corrected for blood absorbance. This can either be done by
normalizing the discriminant factor to blood reflectance. P corr
= P _ R blood Equation .times. .times. 5 Or by
subtracting the blood reflectance. P.sub.corr=P-d.R.sub.blood Equation 6
Where d is an empirical correction factor and R.sub.blood is the
reflectance of blood at an empirically selected wavelength.

[0124] 6. Alternatively the intensity set, F.sub..lamda.m, R.sub..lamda.m
and R.sub..lamda.x, where .lamda. is selected for each fluorophore are
collectively modulated against the pathology results in a principle
component analysis or a logistic regression. These can then form the
basis of pattern recognition techniques, such as, for example,
classification and regression trees (CART), as taught by L. Brieman, et
al. in Classification and Regression Trees, Monterey Calif.: Wadsworth &
Brooks/Cole, 1984, which is hereby incorporated by reference, normal
networks and hybrids thereof.

[0125] The techniques according to the invention are designed to
discriminate normal tissue from various cancerous tissue stages based on
spectroscopic data alone. Additional factors, such, as for example,
patient age, menopausal status, menstrual state, previous history of
disease can be added to the spectroscopic input in achieving better
discrimination.

[0126] In each of the embodiments described above, in which a plurality of
measurement cycles are conducted on a target tissue, and an inner core
having optical fibers arranged in a predetermined pattern is rotated
between measurement cycles to make a plurality of measurements on a
target tissue, alternate embodiments could use some other movement
mechanism other than a rotating one. The invention encompasses other
types of movement or translational devices that allow a plurality of
measurements to be taken on a target tissue with a limited number of
detectors that are spaced far enough apart to avoid cross-talk. Also, as
previously discussed, the measurements could be taken over the entire
area of the subject tissue simultaneously, or the target tissue could be
divided into field areas and measurements could be taken in each field
area.

[0127] Further, the apparatus and methods embodying the invention make it
possible to conduct in vivo measurements of tissues on the inside of body
passages or lumens. An endoscope embodying the invention can be inserted
into a natural body lumen of a human or animal to search for the presence
of cancerous or diseased tissue. This means that no surgery would be
required to locate and examine tissues inside the body of the human or
animal under study.

[0128] The use together of fluorescence measurements along with
reflectance measurements provides a more accurate determination of target
tissue characteristics than one of the measurements alone.

[0129] The techniques described above can be used to map the conditions of
an area of target tissue. For instance, the above-described techniques
can be used to determine a condition of a target tissue adjacent a distal
end of a measuring device. The measuring device could then be moved
adjacent a different portion of the target tissue, and the measurements
could be repeated. This process could be repeated numerous times to
determine the conditions of different portions of a target tissue area.
The determined conditions could then be used to create a map of the
target tissue area, which could be printed or displayed on a monitor.

[0130] One of the most difficult problems with in vivo tissue diagnostics
and disease measurement is the biological diversity of normal tissue
properties between different patients, or even within the same patient.
Furthermore, this diversity is time variant both in the long term and in
the short term. Long term variations may be due to patient age, hormonal
milieu, metabolism, mucosal viscosity, and circulatory and nervous system
differences. Short term variations may be from blood perfusion changes
due to heart beat, physical movement, local temperature changes etc.

[0131] Because of the variability of tissue characteristics, to accurately
determine whether a target tissue is diseased, one needs to compare
measurements of the target tissue to measurements of normal tissues from
the same patient. The measurements of the known normal tissue should be
made concurrently or simultaneously with the measurements of the target
tissue. The normal tissue measurements then serve as a baseline for
normalcy, variations from which may be interpreted as disease. To arrive
at a baseline measurement, a number of strategies can be used.

[0132] First, visual characteristics such as pigmentations (nevi) in skin,
or polyps in the colon, can be used to identify potentially abnormal
regions. Normalized or averaged spectra of multiple regions surrounding
these potentially abnormal, visually distinct regions can be used to
establish baseline measurements. The baseline measurements can then be
compared to measurements taken on the abnormal, visually distinct
regions.

[0133] Measurements of normal and abnormal regions based on visual
characteristics could be automated using imaging capabilities of the
measurement device itself.

[0134] In an alternate strategy, measurements can be taken on spaced apart
regions along a portion of a lumen or tissue. The spacing between the
regions would be dependent on the type of tissue being diagnosed. Then,
differentials between individual measurements taken at different regions
would be calculated. If differentials are greater than a preset amount,
the tissue between the excessively high differentials would be diagnosed
as diseased. In yet another alternate strategy, a gradient in spectral
response as one moves away from a visually suspicious site could also be
used as a marker for disease. This is easily automated and can be
implemented effectively in any imaging modality.

[0135] In addition, pattern recognition algorithms (e.g. neural nets)
could also be used to analyze differences in readings taken from various
sites in the same patient or from multiple readings from different
patients.

[0136] Preliminary testing was completed utilizing the above taught
apparatus and methods to determine the effectiveness of the invention in
determining tissue changes in the cervix. The results are set forth
below. The testing compared the results obtained by the invention to
cytology, colopscopy and histology results.

[0137] The study involved 27 human enrollees; however, data from one
patient could not be collected due to an equipment error. Five of the
patients were measured using a first generation probe having a rigid
transparent window at the device/cervix interface and a monochrometer
capable of producing excitation electromagnetic radiation at wavelengths
of 290 and 460 mm. However, signal-to-noise analyses indicated that stray
light and other problems rendered much of the data unusable, especially
at higher wavelengths (460 nm) with respect to the fluorescence
measurements and at all wavelengths with respect to the reflectance
measurements. For the remaining patients, a second generation probe was
used having a flexible window as well as a new monochrometer, which
allowed an additional excitation electromagnetic wavelength of 350 nm to
be used.

[0139] Of the six high grade dysplasias/cancer, the Pap test
mis-classified three as being either normal, reactive or ASCUS
(sensitivity=50%). Of the ten sub-high grade cases for which both Pap
test and biopsy results were available, the Pap test classified all ten
as sub-high grade (specificity=100%). Colposcopy also classified only
three of six high grade/cancer cases accurately (50% sensitivity) but
correctly classified eight of ten sub-high grade lesions correctly
(specificity=80%).

[0140] Intensities were examined at specific wavelengths which correspond
to the presence and activity of known biomolecules in cervical tissue.
Fluorescence measurements were taken at wavelengths of approximately 290
nm (Tryptophan), 350 nm (NADH) and 460 nm (FAD). Reflectance measurements
were taken at wavelengths of approximately 320 nm, 420 nm (Hemoglobin)
and a range of 540-580 nm (Hemoglobin). These reference the dominant
biomolecules for these wavelengths; however, secondary biomolecules, such
as, for example, collagen and elastin may also be excited. A reflectance
peak was found at about 320 nm which appears to represent a point in the
spectrum where the above discussed and other biomolecules do not absorb,
thus producing the observed reflectance peak.

[0141] The measurements were made using a fiber optic system, which
acquired fluorescence and reflectance intensity data as a series of CCD
images. In order to extract meaningful data, the tissue spectra underwent
a series of correction and calibration operations prior to tissue
measurements. Wavelength calibration was performed, which involves
assigning a wavelength to each spectral data point. Background
subtraction was performed, which involves removing the "dark" signal
present on the CCD and any ambient light signal (e.g. from room lights)
acquired during tissue measurements. Intensity calibration was performed,
which involves normalizing tissue spectral intensities by the intensities
measured of a fluorescence/reflectance standard. Stray light correction
was performed, which involves correcting tissue fluorescence spectra for
excitation monochromator stray light. Also, a system response correction
was performed, which involves correcting for the non-uniform spectral
response of the collection system (optical fibers, filter, spectrograph,
CCD).

[0142] In order to assess data quality, signal-to-noise ratio (SNR), cross
talk and variability were examined. Signal-to-noise ratio is electronic
noise from the CCD as well as optical "noise" and artifacts superimposed
on the fluorescence signal. If the SNR is greater than needed, the
exposure time can be reduced. If the SNR is too low, the exposure time
may need to be increased and other options explored. Cross talk occurs
when the amount of light collected by a given collection optical fiber is
influenced by other surrounding excitation fibers. This parameter is
strongly dependent on the stand off between the fibers and the tissue.
Variability involves the amount of inter- and intra-patient variability
in spectra (e.g., intensity and/or location of peaks) measured from
epithelium in a given state (normal, dysplastic, etc.) which would
influence the diagnostic capability of fluorescence/reflectance-based
discrimination.

[0143] During this testing, three general types of data analysis were
performed: mean, standard deviation and coefficient of variance. The
mean, standard deviation and coefficient of variance of the intensity at
each of the wavelengths for all the spectra from a patient were
calculated. Analyses were performed using measurements taken at 252 data
points distributed over the whole surface of the cervix. The cervix was
also divided into quadrants and measurements were made for each quadrant.
Then, quadrants containing normal tissue biopsy results were compared
with quadrants containing tissue having abnormal biopsy results.

[0144] Inspection of the spectra for each patient from the 252 points on
the cervix would be the most straightforward means of estimating SNR. For
assessing crosstalk and variability, an effective means is to produce
false-color maps of the cervix based on the spectral data. A key
parameter of each spectrum (e.g. intensity at a signal wavelength,
intensity ratio between two wavelengths) can be color coded and mapped to
the location at which it was measured on the cervix. Maps of this type
allow the large volume of data acquired from each patient to be condensed
to a more manageable form for obtaining qualitative insight into spatial
relationships in the data.

[0145] The fluorescence and reflectance intensity measurements, at each of
the respective wavelengths, for all 252 data points on the cervix, was
then analyzed in several different ways. First, the mean, standard
deviation and the coefficient of variance was calculated using the
measurement results from all 252 data points. Graphs depicting these
calculated values appear in FIGS. 13-26. The data points are
characterized as normal, low grade dysplasia/inflammation, or high grade
displasia based on a histological examination that was performed
subsequent to the spectroscopic measurements.

[0146] Next, the cervix was divided into four zones, or field areas, and
mean, standard deviation and coefficient of variance values were
calculated on a zone-by-zone basis. The results for each zone can then be
compared to one another to attempt to localize potentially abnormal zones
on the cervix. The calculated values for each zone were then examined to
determine if a sufficient signal-to-noise ration had been obtained. If
the signal-to-noise ratio for a particular zone was too low, the data for
that zone was discarded.

[0147] Also, as mentioned above, a subsequent histological examination was
performed on the tissue samples collected from each patient. If a tissue
sample was taken and analyzed for a particular zone having the requisite
signal-to-noise ratio, the result is plotted in FIGS. 27-38. However, if
a tissue sample for a particular zone of a patient was not obtained and
analyzed, there was no way to characterize the data point, and the
results were not plotted in FIGS. 27-38. Thus, the data points in FIGS.
27-38 only represent quadrants that had a sufficiently high
signal-to-noise ratio, and that were subsequently histologically analyzed
to determine their actual condition.

[0148] Finally, the data was analyzed on a "by-rotation basis." As
described above, the device used to collect the data has forty-two (42)
optical fiber interrogation points distributed over the face that
contacts the cervix. A first measurement cycle is conducted to collect 42
measurement results. Then, the optical fibers are rotated 60.degree..
During a second measurement cycle, an additional 42 measurement results
are obtained at the new locations. This process of rotation and
measurement is repeated until measurements have been conducted at all 252
points across the cervix.

[0149] The obtained measurements were analyzed on a by-rotation basis. In
other words, the mean, standard deviation and coefficient of variance was
calculated for the 42 measurements taken during the first measurement
cycle, the new values were calculated using the 42 measurements taken
during each subsequent measurement cycle. Note, that the measurement
results from each cycle are substantially evenly distributed over the
entire cervix. All the calculated values for each by-rotation measurement
cycle are shown in FIGS. 39-50.

[0150] FIGS. 13 and 14 show biparameter plots of the means versus the
coefficient of variance (CV) and standard deviation (SD), respectively,
for all twenty-one cases. The first five cases were standardized to take
into account the differences in window type between these and the other
sixteen cases. In FIG. 13, the six high grade/cancer cases appear to be
clustered in the upper right hand corner of the graph (above the diagonal
line and to the right of the three low grade lesions above the line).

[0151] The remaining analyses involved the latter sixteen patients tested
with the second generation probe and new monochrometer. FIGS. 15-26 show
the calculated values for the whole cervix. Table 2 below summarizes the
degree of overlap between high grade cases and low
grade/inflammation/normal cases for each of the three fluorescence and
reflectance measurements, showing the percentage of correct negative
predictions where n=12 using the threshold below the lowest level measure
for the four high grade cases (i.e., at 100% sensitivity). Table 2
includes all sub-high grade dysplasia case, including low grade dysplasia
(n=7), inflammation (n=3), abnormal Pap results but no biopsy (n=2),
symptomless patient with history of disease who underwent an experimental
treatment (n=2), and normals (n=1). As can be seen in Table 2,
wavelengths of 290 nm for fluorescence measurements and 320 nm for
reflectance measurements show the least amount of overlap between high
grade and sub-high grade cases.
TABLE-US-00002
TABLE 2
Standard Coefficient of
Variable Mean Deviation Variance
290 FL Excitation 58% 75% 75%
350 FL Excitation 42% 17% 58%
460 FL Excitation 67% 0% 33%
320 Reflectance 25% 67% 75%
420 Reflectance 8% 67% 58%
540-580 Reflectance 25% 58% 67%

[0152] FIGS. 27-38 show the calculated measurements for quadrants that had
a sufficiently high signal-to-noise ration, and that were subsequently
analyzed to determine their condition. The objective of the by quadrant
analysis was to indicate whether spatial information down to the quadrant
level was available and to determine whether normal quadrants could be
differentiated from abnormal quadrants.

[0153] A total of nineteen quadrants could be reliably identified as
containing either a diseased or normal biopsied site. Of these, none were
normal quadrants, eight contained low grade/inflammatory disease and two
contained high grade disease. Use of a wavelength of 290 nm for
fluorescence measurements appeared to separate the data by virtue of
within quadrant measures of variability (SD and CV). Mean fluorescence
appears to be discriminative at 350 and 420 nm. There appears to one high
grade lesion, which was diagnosed by Pap test as normal and by colposcopy
as metaplasia, which can be misdiagnosed as high grade disease at biopsy.
Thus, while it was seen previously that whole cervix measurements were of
little diagnostic value, when taken down to the quadrant level, i.e., a
smaller field area, the measurements become more diagnosticly
"meaningful".

[0154] FIGS. 39-50 show the by rotation calculated measurements. This
analysis was done in order to determine whether individual rotation data,
from a single measurement cycle, provided any clue as to whether all or a
subset for the six rotation positions are necessary. In general, the
single rotation data mirror that of the integrated data set, with a bit
more overlap. Based on the results, it appears that the by-rotation data
is similar to the entire cervix data. This suggests that the resolution
obtained from 42 interrogation points may be sufficient to accurately
predict the condition of the cervix.

[0155] FIG. 51 shows a biparameter plot of the calculated standard
deviation of the reflectance measurements using a wavelength of 320 nm
against the calculated means of the fluorescence measurements using a
wavelength of 460 nm. As can be seen, this plot show a differentiation
between normal and high grade lesions.

[0156] Although it is premature to draw definitive conclusions regarding
this small data set, the result are encouraging. There were high grade
lesions misclassified by both Pap tests and colposcopy which could be
discriminated by the spectroscopic methods of the invention. Moreover,
the results of these preliminary cases are consistent with known biologic
phenomena and field effects due to carcinogenesis. Of note is that both
fluorescence and reflectance measurements provide discriminative
information.

[0157] Having looked at overall means, standard deviation and coefficient
of variation at individual wavelengths, spatial and spectral information
can then be exploited. Those spectra measured from points on the tissue
for which histopathology is available (e.g., at/near a biopsy site) can
be examined specifically by category, for example, normal versus
abnormal. To further utilize spectral information, the preferred method
involves taking various intensity ratios at the key wavelengths discussed
above. Beyond that approach, advanced statistical analysis techniques
(for example, principal component analysis, Bayesian Classification,
Classification Trees, Artificial Neural Networks,) may be used to help to
identify other wavelengths which can be effective for discriminating and
modeling a pattern recognition.

[0158] The foregoing embodiments are merely exemplary and are not to be
construed as limiting the invention. The present teaching can be readily
applied to other types of apparatuses. The description of the invention
is intended to be illustrative, and not to limit the scope of the claims.
Many alternatives, modifications, and variations will be apparent to
those skilled in the art.